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Summary: Analyses of long non-coding RNA (lncRNA)-protein interactions provide key clues for understanding the molecular basis of lncRNA-modulated biological processes. Here, we detail a yeast three-hybrid assay to identify the lncRNA-interacting protein. We describe steps for lncRNA bait preparation, screening an RNA-binding proteins (RBPs) cDNA library, and validation of the lncRNA-RBP interaction. The assay can also be further applied to delineate the region of RBP that mediates the RNA-protein interaction.For complete details on the use and execution of this protocol, please refer to Zhang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Recurrent implantation failure (RIF) presents a significant clinical challenge due to the lack of established diagnostic and therapeutic guidelines. Emerging evidence underscores the crucial role of competitive endogenous RNA (ceRNA) regulatory networks in non-cancerous female reproductive disorders, yet the intricacies and operational characteristics of these networks in RIF are not fully understood. This study aims to demystify the ceRNA regulatory network and identify potential biomarkers for its diagnosis. We analyzed expression profiles of three RNA types (long noncoding RNAs [lncRNAs], microRNAs [miRNAs], and mRNAs) sourced from the GEO database, leading to the identification of the H19-hsa-miR-301a-3p-GAS1 ceRNA network. This network demonstrates significant diagnostic relevance for RIF. Notably, the H19/GAS1 axis within this ceRNA network, identified through correlation analysis, emerged as a promising diagnostic marker, as evidenced by operating receiver operator characteristic (ROC) curve analysis. Further investigation into the binding potential of miR-301a-3p with H19 and GAS1 revealed a close association of these genes with endometrial disorders and embryo loss, as per the Comparative Toxicogenomics Database. Additionally, our immune infiltration analysis revealed a lower proportion of T cells gamma delta (γδ) in RIF, along with distinct differences in the expression of immune cell type-specific markers between fertile patients and those with RIF. We also observed a correlation between aberrant expression of H19/GAS1 and these immune markers, suggesting that the H19/GAS1 axis might play a role in modifying the immune microenvironment, contributing to the pathogenesis of RIF. In conclusion, the ceRNA-based H19/GAS1 axis holds promise as a novel diagnostic biomarker for RIF, potentially enhancing our understanding of its underlying mechanisms and improving the success rates of implantation. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is an incurable hematological malignancy with poor survival. Accumulating evidence reveals that lactylation modification plays a vital role in tumorigenesis. However, research on lactylation-related genes (LRGs) in predicting the prognosis of MM remains limited. Differentially expressed LRGs (DELRGs) between MM and normal samples were investigated from the Gene Expression Omnibus database. Univariate Cox regression and LASSO Cox regression analysis were applied to construct gene signature associated with overall survival. The signature was validated in two external datasets. A nomogram was further constructed and evaluated. Additionally, Enrichment analysis, immune analysis, and drug chemosensitivity analysis between the two groups were investigated. qPCR and immunofluorescence staining were performed to validate the expression and localization of PFN1. CCK-8 and flow cytometry were performed to validate biological function. A total of 9 LRGs (TRIM28, PPIA, SOD1, RRP1B, IARS2, RB1, PFN1, PRCC, and FABP5) were selected to establish the prognostic signature. Kaplan–Meier survival curves showed that high-risk group patients had a remarkably worse prognosis in the training and validation cohorts. A nomogram was constructed based on LRGs signature and clinical characteristics, and showed excellent predictive power by calibration curve and C-index. Moreover, biological pathways, immunologic status, as well as sensitivity to chemotherapy drugs were different between high- and low-risk groups. Additionally, the hub gene PFN1 is highly expressed in MM, knocking down PFN1 induces cell cycle arrest, suppresses cell proliferation and promotes cell apoptosis. In conclusion, our study revealed that LRGs signature is a promising biomarker for MM that can effectively early distinguish high-risk patients and predict prognosis. [ABSTRACT FROM AUTHOR]

Background: The effect of oxycodone as an opioid receptor agonist on immune function is still controversial. In this study, we investigated the possible effects of oxycodone on immune function in mice and its possible mechanisms of action. Methods: By repeated intraperitoneal injections of 25 mg/kg morphine and 5 mg/kg, 20 mg/kg, and 60 mg/kg oxycodone, we assessed possible changes in the number of splenic lymphocytes and inflammatory cytokines in the serum of mice. CD4+ T cells and CD8+ T cells were sorted from the spleen to observe whether the expression levels of opioid receptors and downstream signals were altered. Results: Repeated administration of oxycodone at a dose above 20 mg/kg resulted in significant weight loss. Repeated administration of oxycodone exhibits significant dose-dependent reduction in CD4+ T cells, with little effect on CD8+ T cells and little effect on inflammatory cytokine levels. Low- and intermediate-dose oxycodone increased the mRNA expression level of MOR, KOR, and DOR to varying degrees. Moreover, oxycodone increases the mRNA expression levels of the TLR4 signaling pathway to varying degrees. Conclusion: Repeated intraperitoneal injection of oxycodone induces immunosuppression in mice. [ABSTRACT FROM AUTHOR]

Anthocyanins and proanthocyanidins are considered to be essential secondary metabolites in grapes and are used to regulate metabolic processes, while miRNAs are involved in their synthesis of anthocyanins and proanthocyanidins to regulate metabolic processes. The present research work was carried out to investigate the underlying regulatory mechanism of target genes in the grape cultivars 'Italia' and 'Benitaka'. miRNA and transnscriptomic sequencing technology were employed to characterize both the profiles of miRNAs and the transcripts of grape peels at 10 and 11 weeks post flowering (10 wpf and 11 wpf). The results revealed that the expression level of vvi-miR828a in 'Italia' at 10 and 11 wpf was significantly higher than that in 'Benitaka'. miRNA-seq analysis predicted MYBPA1 to be the target gene of vvi-miR828a. In transcriptome analysis, the expression level of the VvMYBPA1 gene in 'Benitaka' was significantly higher than that in 'Italia'; in addition, the TPM values (expression levels) of VvMYBPA1 and miR828a also showed an evident negative correlation. The determination of the proanthocyanidin (PA) content in 'Italia' and 'Benitaka' peels at 11 wpf demonstrated that the PA content of 'Benitaka' was significantly higher than that of 'Italia'. The outcomes of RT-qRCR analysis exhibited that the expression levels of the VdPAL, VdCHS, VdCHI, VdDFR, VdMYB5b, VdANR, and VdMYBPA1 genes related anthocyanin and proanthocyanidin pathways were reduced, while the expression levels of all of the above genes were increased after the transient expression of the VvMYBPA1 vector into grape leaves. The results of the transient overexpression experiment of vvi-miR828a before the veraison period of strawberry fruits showed that vvi-miR828a can significantly slow down the coloration of strawberries. The vvi-miR828a negatively regulates the accumulation of proanthocyanidins in grape fruits by inhibiting the expression of VvMYBPA1. [ABSTRACT FROM AUTHOR]

Introduction The number of smokers worldwide increased greatly during the past decades and reached 1.14 billion in 2019, becoming a leading risk factor for human health. Tobacco smoking has wide effects on human genetics, epigenetics, transcriptome, and gut microbiome. Although many studies have revealed effects of smoking on host transcriptome, research on the relationship between smoking, host gene expression, and the gut microbiome is limited. Aims and Methods We first explored transcriptome and metagenome profile differences between smokers and nonsmokers. To evaluate the relationship between host gene expression and gut microbiome, we then applied bidirectional mediation analysis to infer causal relationships between smoking, gene expression, and gut microbes. Results Metagenome and transcriptome analyses revealed 71 differential species and 324 differential expressed genes between smokers and nonsmokers. With smoking as an exposure variable, we identified 272 significant causal relationships between gene expression and gut microbes, among which there were 247 genes that mediate the effect of smoking on gut microbes. Pathway-based enrichment analysis showed that these genes were significantly enriched in heme metabolic pathway, which mainly mediated the changes of Bacteroides finegoldii and Lachnospiraceae bacterium 9_1_43BFAA. Additionally, by performing metabolome data analysis in the Integrated Human Microbiome Project (iHMP) database, we verified the correlation between the intermediate products of the heme metabolism pathway (porphobilinogen, bilirubin, and biliverdin) and gut microbiome. Conclusions By investigating the bidirectional interaction between smoking-related host gene expression and gut microbes, this study provided evidence for the mediation of smoking on gut microbes through co-involvement or interaction of heme metabolism. Implications By comparing the metagenome and transcriptome sequencing profiles between 34 smokers and 33 age- and gender-matched nonsmokers, we are the first to reveal causal relationships among tobacco smoking, host gene expression, and gut microbes. These findings offer insight into how smoking affects gut microbes through host gene expression and metabolism, which highlights the importance of heme metabolism in modulating the effects of smoking on gut microbiome. [ABSTRACT FROM AUTHOR]

Background: Transcriptome-wide association study (TWAS) is an influential tool for identifying genes associated with complex diseases whose genetic effects are likely mediated through transcriptome. TWAS utilizes reference genetic and transcriptomic data to estimate effect sizes of genetic variants on gene expression (i.e., effect sizes of a broad sense of expression quantitative trait loci, eQTL). These estimated effect sizes are employed as variant weights in gene-based association tests, facilitating the mapping of risk genes with genome-wide association study (GWAS) data. However, most existing TWAS of Alzheimer's disease (AD) dementia are limited to studying only cis-eQTL proximal to the test gene. To overcome this limitation, we applied the Bayesian Genome-wide TWAS (BGW-TWAS) method to leveraging both cis- and trans- eQTL of brain and blood tissues, in order to enhance mapping risk genes for AD dementia. Methods: We first applied BGW-TWAS to the Genotype-Tissue Expression (GTEx) V8 dataset to estimate cis- and trans- eQTL effect sizes of the prefrontal cortex, cortex, and whole blood tissues. Estimated eQTL effect sizes were integrated with the summary data of the most recent GWAS of AD dementia to obtain BGW-TWAS (i.e., gene-based association test) p-values of AD dementia per gene per tissue type. Then we used the aggregated Cauchy association test to combine TWAS p-values across three tissues to obtain omnibus TWAS p-values per gene. Results: We identified 85 significant genes in prefrontal cortex, 82 in cortex, and 76 in whole blood that were significantly associated with AD dementia. By combining BGW-TWAS p-values across these three tissues, we obtained 141 significant risk genes including 34 genes primarily due to trans-eQTL and 35 mapped risk genes in GWAS Catalog. With these 141 significant risk genes, we detected functional clusters comprised of both known mapped GWAS risk genes of AD in GWAS Catalog and our identified TWAS risk genes by protein-protein interaction network analysis, as well as several enriched phenotypes related to AD. Conclusion: We applied BGW-TWAS and aggregated Cauchy test methods to integrate both cis- and trans- eQTL data of brain and blood tissues with GWAS summary data, identifying 141 TWAS risk genes of AD dementia. These identified risk genes provide novel insights into the underlying biological mechanisms of AD dementia and potential gene targets for therapeutics development. [ABSTRACT FROM AUTHOR]

Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production. However, promoters in nonconventional yeasts such as Ogataea polymorpha are always limited, which results in incompatible gene modulation. Here, we expanded the promoter library in O. polymorpha based on transcriptional data, among which 13 constitutive promoters had the strengths ranging from 0-55% of PGAP, the commonly used strong constitutive promoter, and 2 were growth phase-dependent promoters. Subsequently, 2 hybrid growth phase-dependent promoters were constructed and characterized, which had 2-fold higher activities. Finally, promoter engineering was applied to precisely regulate cellular metabolism for efficient production of ß-elemene. The glyceraldehyde-3-phosphate dehydrogenase gene GAP was downregulated to drive more flux into pentose phosphate pathway (PPP) and then to enhance the supply of acetyl-CoA by using phosphoketolasephosphotransacetylase (PK-PTA) pathway. Coupled with the phase-dependent expression of synthase module (ERG20~LsLTC2 fusion), the highest titer of 5.24 g/L with a yield of 0.037 g/(g glucose) was achieved in strain YY150U under fed-batch fermentation in shake flasks. This work characterized and engineered a series of promoters, that can be used to fine-tune genes for constructing efficient yeast cell factories. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to determine the effects of iRoot BP Plus on receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis in vitro and inflammation‐mediated bone resorption in vivo and investigated the underlying molecular mechanisms. Methodology: CCK‐8 was performed to test cell viability in RANKL‐induced RAW 264.7 cells and BMDMs in response to iRoot BP Plus. The effect of iRoot BP Plus on osteoclastogenesis was determined using TRAP staining and phalloidin staining, respectively. Pit formation assay was conducted to measure osteoclast resorptive capacity. Western blot and qPCR were performed to examine osteoclast‐related proteins and gene expression, respectively. Western blot was also used to investigate the signalling pathways involved. For in vivo experiments, an LPS‐induced mouse calvarial bone resorption model was established to analyse the effect of iRoot BP Plus on bone resorption (n = 6 per group). At 7 days, mouse calvaria were collected and prepared for histological analysis. Results: We identified that iRoot BP Plus extracts significantly attenuated RANKL‐induced osteoclastogenesis, reduced sealing zone formation, restrained osteolytic capacity and decreased osteoclast‐specific gene expression (p <.01). Mechanistically, iRoot BP Plus extracts reduced TRAF6 via proteasomal degradation, then suppressed the phosphorylation of mitogen‐activated protein kinases (MAPKs), blocked the nuclear translocation of c‐Fos and diminished nuclear factor‐κB (NF‐κB) p65 and NFATc1 accumulation. Consistent with the in vitro results, iRoot BP Plus extracts attenuated osteoclast activity thus protecting against inflammatory bone resorption in vivo (p <.05), which was accompanied by a suppression of TRAF6, c‐Fos, NFATc1 and cathepsin K expression. Conclusion: These findings provide valuable insights into the signalling mechanisms underlying nanoparticulate bioceramic putty‐mediated bone homeostasis. [ABSTRACT FROM AUTHOR]

SUMMARY: The petals of rose (Rosa sp.) flowers determine the ornamental and industrial worth of this species. The number of petals in roses was previously shown to be subject to fluctuations in ambient temperature. However, the mechanisms by which rose detects and responds to temperature changes are not entirely understood. In this study, we identified short interstitial telomere motifs (telo boxes) in the second intron of AGAMOUS (RcAG) from China rose (Rosa chinensis) that play an essential role in precise temperature perception. The second intron of RcAG harbors two telo boxes that recruit telomere repeat binding factors (RcTRBs), which interact with CURLY LEAF (RcCLF) to compose a repressor complex. We show that this complex suppresses RcAG expression when plants are subjected to low temperatures via depositing H3K27me3 marks (trimethylation of lysine 27 on histone H3) over the RcAG gene body. This regulatory mechanism explains the low‐temperature‐dependent decrease in RcAG transcript levels, leading to the production of more petals under these conditions. Our results underscore an interesting intron‐mediated regulatory mechanism governing RcAG expression, enabling rose plants to perceive temperature cues and establish petal numbers. Significance Statement: Our research underscores a new intron‐mediated regulatory mechanism governing RcAG expression, enabling rose plants to perceive temperature cues and establish petal numbers. It might be of great interest to a broad range of readers including environmental response, epigenetic modification, floral organ development, and other researchers in related fields. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the effect of subgingival delivery of progranulin (PGRN)/gelatin methacryloyl (GelMA) complex as an adjunct to scaling and root planing (SRP) on an experimental periodontitis dog model with Class II furcation involvement (FI). Materials and Methods: A Class II FI model was established, and the defects were divided into four treatment groups: (a) no treatment (control); (b) SRP; (c) SRP + GelMA; (d) SRP + PGRN/GelMA. Eight weeks after treatment, periodontal parameters were recorded, gingival crevicular fluid and gingival tissue were collected for ELISA and RT‐qPCR, respectively, and mandibular tissue blocks were collected for micro computed tomography (micro‐CT) scanning and hematoxylin and eosin (H&E) staining. Results: The SRP + PGRN/GelMA group showed significant improvement in all periodontal parameters compared with those in the other groups. The expression of markers related to M1 macrophage and Th17 cell significantly decreased, and the expression of markers related to M2 macrophage and Treg cell significantly increased in the SRP + PGRN/GelMA group compared with those in the other groups. The volume, quality and area of new bone and the length of new cementum in the root furcation defects of the PGRN/GelMA group were significantly increased compared to those in the other groups. Conclusions: Subgingival delivery of the PGRN/GelMA complex could be a promising non‐surgical adjunctive therapy for anti‐inflammation, immunomodulation and periodontal regeneration. [ABSTRACT FROM AUTHOR]

Tumor microenvironment (TME) is a complex dynamic system with many tumor-interacting components including tumor-infiltrating leukocytes (TILs), cancer associated fibroblasts, blood vessels, and other stromal constituents. It intrinsically affects tumor development and pharmacology of oncology therapeutics, particularly immune-oncology (IO) treatments. Accurate measurement of TME is therefore of great importance for understanding the tumor immunity, identifying IO treatment mechanisms, developing predictive biomarkers, and ultimately, improving the treatment of cancer. Here, we introduce a mouse-IO NGS-based (NGSmIO) assay for accurately detecting and quantifying the mRNA expression of 1080 TME related genes in mouse tumor models. The NGSmIO panel was shown to be superior to the commonly used microarray approach by hosting 300 more relevant genes to better characterize various lineage of immune cells, exhibits improved mRNA and protein expression correlation to flow cytometry, shows stronger correlation with mRNA expression than RNAseq with 10x higher sequencing depth, and demonstrates higher sensitivity in measuring low-expressed genes. We describe two studies; firstly, detecting the pharmacodynamic change of interferon-γ expression levels upon anti-PD-1: anti-CD4 combination treatment in MC38 and Hepa 1–6 tumors; and secondly, benchmarking baseline TILs in 14 syngeneic tumors using transcript level expression of lineage specific genes, which demonstrate effective and robust applications of the NGSmIO panel. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is the most common form of dementia in the elderly marked by central nervous system (CNS) neuronal loss and amyloid plaques. FAM222A, encoding an amyloid plaque core protein, is an AD brain atrophy susceptibility gene that mediates amyloid-β aggregation. However, the expression interplay between FAM222A and other AD-related pathway genes is unclear. Objective: Our goal was to study FAM222A's whole-genome co-expression profile in multiple tissues and investigate its interplay with other AD-related genes. Methods: We analyzed gene expression correlations in Genotype-Tissue Expression (GTEx) tissues to identify FAM222A co-expressed genes and performed functional enrichment analysis on identified genes in CNS system. Results: Genome-wide gene expression profiling identified 673 genes significantly correlated with FAM222A (p < 2.5×10–6) in 48 human tissues, including 298 from 13 CNS tissues. Functional enrichment analysis revealed that FAM222A co-expressed CNS genes were enriched in multiple AD-related pathways. Gene co-expression network analysis for identified genes in each brain region predicted other disease associated genes with similar biological function. Furthermore, co-expression of 25 out of 31 AD-related pathways genes with FAM222A was replicated in brain samples from 107 aged subjects from the Aging, Dementia and TBI Study. Conclusion: This gene co-expression study identified multiple AD-related genes that are associated with FAM222A, indicating that FAM222A and AD-associated genes can be active simultaneously in similar biological processes, providing evidence that supports the association of FAM222A with AD. [ABSTRACT FROM AUTHOR]

The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late geneCG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of themigrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells. [ABSTRACT FROM AUTHOR]

SUMMARY: Cannabis glandular trichomes (GTs) are economically and biotechnologically important structures that have a remarkable morphology and capacity to produce, store, and secrete diverse classes of secondary metabolites. However, our understanding of the developmental changes and the underlying molecular processes involved in cannabis GT development is limited. In this study, we developed Cannabis Glandular Trichome Detection Model (CGTDM), a deep learning‐based model capable of differentiating and quantifying three types of cannabis GTs with a high degree of efficiency and accuracy. By profiling at eight different time points, we captured dynamic changes in gene expression, phenotypes, and metabolic processes associated with GT development. By integrating weighted gene co‐expression network analysis with CGTDM measurements, we established correlations between phenotypic variations in GT traits and the global transcriptome profiles across the developmental gradient. Notably, we identified a module containing methyl jasmonate (MeJA)‐responsive genes that significantly correlated with stalked GT density and cannabinoid content during development, suggesting the existence of a MeJA‐mediated GT formation pathway. Our findings were further supported by the successful promotion of GT development in cannabis through exogenous MeJA treatment. Importantly, we have identified CsMYC4 as a key transcription factor that positively regulates GT formation via MeJA signaling in cannabis. These findings provide novel tools for GT detection and counting, as well as valuable information for understanding the molecular regulatory mechanism of GT formation, which has the potential to facilitate the molecular breeding, targeted engineering, informed harvest timing, and manipulation of cannabinoid production. [ABSTRACT FROM AUTHOR]

Introduction: The aim of this study was to investigate the effect and mechanism of kaempferol on alcoholic steatohepatitis. Methods: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for 6 weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1β, and TNF-α were detected by kits; lipopolysaccharide (LPS) expression was detected by ELISA kit, and the expression of IL-1β and TNF-α was assessed by qRT-PCR; Western blot was utilized to assess the excessive inflammatory response of liver and colon tissue and the related signaling pathway activation. Results: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1β and tumor necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their cytokine suppressor signaling 1 (SOCS1) protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and zonula occludens-1. More importantly, kaempferol significantly reduced serum LPS levels in alcoholic steatohepatitis mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. Conclusion: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine. [ABSTRACT FROM AUTHOR]

Grassland plants that endure livestock grazing exhibit a dwarf phenotype, which can be transmitted to clonal offspring. Yet to date, it remains poorly understood whether such transgenerational dwarf effects alter the plants' response to shade. Here, we conducted a common garden experiment under sunlight and shade conditions with clonal Leymus chinensis offspring, the parents of which had endured livestock overgrazing (OG) and non-grazing (NG) in the field, respectively. Plant morphological, physiological, and transcriptomic analyses were carried out. The results indicated that NG offspring showed greater shade avoidance than OG offspring. That is, NG offspring exhibited greater plasticity of vegetative height and leaf width, which may be contributed to their greater photosynthetic capacity and gibberellin (GA3) content compared with OG offspring when treated with shade. In addition, RNA-Seq profiling showed that differentially expressed genes in NG offspring were mainly enriched in RNA modification and metabolic processes, which facilitated rapid response to shade. Phytochrome interacting factors (PIFs) promoted downstream shade marker genes in NG offspring by significantly downregulating the expression of PHYC, SPY, and DELLA. Our findings suggest that light conditions should be taken into account to better understand transgenerational dwarf effects induced by livestock grazing on grassland ecosystems. These results provide new insights into the inducible factors of phenotypic variations in grassland plants that experience grazing. [ABSTRACT FROM AUTHOR]

Homeodomain-Leucine Zipper (HD-Zip) proteins are important ubiquitous and diverse molecular chaperones in plants. We characterized a gene CaATHB-12 derived from HD-Zip I subfamily which was intensively induced by exogenous abscisic acid (ABA), salt, and mannitol applications in a pepper cultivar. Efficient gene silencing lines were created from pepper, and stable heterologous overexpression lines were created from Arabidopsis to achieve a comprehensive exploration of gene function. The functional study of CaATHB-12 in pepper increased plant sensitivity to ABA stress, while the over-expressing CaATHB-12 in Arabidopsis lines revealed that tolerance to ABA, salt, and mannitol stresses was decreased. Furthermore, CaATHB-12 plays a fundamental role in elevating the tolerance to these stresses through the increased expression of other stress related genes, increasing the activities of antioxidant enzymes and scavenging the reactive oxygen species. The studied functions of the CaATHB-12 gene may provide some insights in exquisite molecular detail by pursuing signal transduction mechanisms that converge on gene expression patterns. [ABSTRACT FROM AUTHOR]

Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy threatening patients' life quality. Our previous study has demonstrated that inhibition of long non-coding RNA H19 (lncRNA h19; H19) blocks ATC growth and metastasis. The current study aimed to further explore the potential mechanism of H19 in ATC. Expression of H19, miR-454-3p, and BHLHE40 mRNA was measured using RT-qPCR in tissue samples and cell lines. The dual-luciferase reporter assay and Pearson correlation analysis were used to explore the interaction among H19, miR-454-3p, and BHLHE40. The biological process of proliferation, migration, and invasion was determined using loss- or gain-function CCK-8 and Transwell assays. Western blot assay was used to evaluate the changes in protein levels. H19 was elevated in ATC tissues and cell lines. Based on online prediction database results, miR-454-3p might be a target of H19, and BHLHE40 might be a direct target of miR-454-3p. miR-454-3p expression was decreased in ATC and had a negative interaction with H19. BHLHE40 mRNA expression was increased and has a negative correlation with miR-454-3p and a positive correlation with H19. Downregulation of miR-454-3p and upregulation of BHLHE40 could reverse the decreased cellular activities caused by si-H19. Moreover, the silence of H19 modulates BHLHE40 to affect the PI3K/AKT protein levels and apoptotic-related protein levels. The current study provided a potential detailed mechanism of H19 in ATC, and lncRNA H19-miR-454-3p-BHLHE40 interaction may be a new experimental basis for prognosis and targeted therapy for ATC patients. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is a life‐threatening condition characterized by lung inflammation and high mortality rates. Lung cancer, specifically lung adenocarcinoma (LUAD), is a major cause of cancer‐related deaths worldwide. Patients with LUAD, particularly those undergoing chemotherapy, are more likely to develop ARDS. ARDS inflicts major malfunctioning in the immune system. We suspected a certain shared pathogenic mechanism between these diseases. This study analyzed 503 LUAD patients from the TCGA‐LUAD cohort as the training set, 85 LUAD cases from the GSE30219 cohort as the validation set, and 24 RNA‐seq samples from ARDS mice model and control groups in the GSE2411 cohort. The differentially expressed genes (DEGs) of ARDS were analyzed using the limma package and screened by Cox and Lasso analysis. ssGSEA and xCell algorithms were utilized for immune landscaping. RT‐qPCR analysis was used to determine the mRNA levels of key genes in both the LPS‐induced ARDS model and human LUAD cell lines. We identified DEGs between ARDS and control groups, which were highly associated with cytokine production and leukocyte migration. A prognosis model for LUAD patients was developed based on the expressions of the key genes in the ARDS‐derived DEGs, including FMO3, IL1R2, CCL20, CFTR, and GADD45G. A satisfactory efficacy was observed in both the training and validation cohorts. The model demonstrated increased effectiveness in predicting the intratumor immune profile and mutation status of LUAD. Moreover, we utilized LPS to induce the ARDS model, which resulted in elevated expressions of IL1R2 and CCL20. Additionally, CCL20 was upregulated in cancerous LUAD cell lines. We developed an ARDS‐based model for stratifying LUAD prognosis. CCL20 was found to be elevated in both the ARDS model and LUAD, suggesting a shared underlying mechanism of these two diseases. [ABSTRACT FROM AUTHOR]

Background: Diabetic cardiomyopathy (DCM) is a serious health-threatening complication of diabetes mellitus characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are a potential therapeutic tool for DCM and myocardial fibrosis via mechanisms such as the regulation of microRNA (miRNA) expression and inflammation. It remains unclear, however, whether hUC-MSC therapy has beneficial effects on cardiac function following different durations of diabetes and which mechanistic aspects of DCM are modulated by hUC-MSC administration at different stages of its development. This study aimed to investigate the therapeutic effects of intravenous administration of hUC-MSCs on DCM following different durations of hyperglycemia in an experimental male model of diabetes and to determine the effects on expression of candidate miRNAs, target mRNA and inflammatory mediators. Methods: A male mouse model of diabetes was induced by multiple low-dose streptozotocin injections. The effects on severity of DCM of intravenous injections of hUC-MSCs and saline two weeks previously were compared at 10 and 18 weeks after diabetes induction. At both time-points, biochemical assays, echocardiography, histopathology, polymerase chain reaction (PCR), immunohistochemistry and enzyme-linked immunosorbent assays (ELISA) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis and expression of fibrosis-related mRNA, miRNA and inflammatory mediators. Results: Saline-treated diabetic male mice had impaired cardiac function and increased cardiac fibrosis after 10 and 18 weeks of diabetes. At both time-points, cardiac dysfunction and fibrosis were improved in hUC-MSC-treated mice. Pro-fibrotic indicators (α-SMA, collagen I, collagen III, Smad3, Smad4) were reduced and anti-fibrotic mediators (FGF-1, miRNA-133a) were increased in hearts of diabetic animals receiving hUC-MSCs compared to saline. Increased blood levels of pro-inflammatory cytokines (IL-6, TNF, IL-1β) and increased cardiac expression of IL-6 were also observed in saline-treated mice and were reduced by hUC-MSCs at both time-points, but to a lesser degree at 18 weeks. Conclusion: Intravenous injection of hUC-MSCs ameliorated key functional and structural features of DCM in male mice with diabetes of shorter and longer duration. Mechanistically, these effects were associated with restoration of intra-myocardial expression of miRNA-133a and its target mRNA COL1AI as well as suppression of systemic and localized inflammatory mediators. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is essential for RNA metabolism in cells. The YTH domain, conserved in the kingdom of Eukaryotes, acts as an m6A reader that binds m6A-containing RNA. In plants, the YTH domain is involved in plant hormone signaling, stress response regulation, RNA stability, translation, and differentiation. However, little is known about the YTH genes in tea-oil tree, which can produce edible oil with high nutritional value. This study aims to identify and characterize the YTH domains within the tea-oil tree (Camellia chekiangoleosa Hu) genome to predict their potential role in development and stress regulation. In this study, 10 members of the YTH family containing the YTH domain named CchYTH1-10 were identified from C. chekiangoleosa. Through analysis of their physical and chemical properties and prediction of subcellular localization, it is known that most family members are located in the nucleus and may have liquid–liquid phase separation. Analysis of cis-acting elements in the CchYTH promoter region revealed that these genes could be closely related to abiotic stress and hormones. The results of expression profiling show that the CchYTH genes were differentially expressed in different tissues, and their expression levels change under drought stress. Overall, these findings could provide a foundation for future research regarding CchYTHs in C. chekiangoleosa and enrich the world in terms of epigenetic mark m6A in forest trees. [ABSTRACT FROM AUTHOR]

Objective: MicroRNA-related single-nucleotide polymorphisms (miR-SNPs) can alter microRNA (miRNA) expression profiles, thereby influencing the risk of rheumatic diseases. Herrin a case control study, six miR-SNPs in miRNA processing machinery genes, namely RAN (rs14035), XPO5 (rs11077), Dicer (rs3742330), GEMIN3 (rs197412), GEMIN4 (rs2740348), and TNRC6B (rs9623117), were genotyped to assess their correlation with the risk of systemic lupus erythematosus (SLE). Methods: We included 119 patients with SLE and 130 healthy controls. The genotypes of the six miR-SNPs were determined using polymerase chain reaction (PCR). Serum cytokine levels were assessed using a cytometric bead array, and fluorescent probe technology was used to determine plasma reactive oxygen species (ROS) levels. Results: The AA genotype of Dicer was correlated with a 0.566-fold decreased risk of SLE compared with that of the AG + GG genotype (odds ratio, 0.566; 95% CI, 0.342–0.935; p =.026), and the rs3742330 A allele was associated with a significantly decreased risk of SLE (p =.035) compared with that of the rs3742330G allele. Additionally, AA genotype carriers exhibited lower levels of interleukin-6 (IL-6) in the blood (p =.013). Subsequent analysis revealed increased ROS production in patients with SLE than that in the controls (621.042 ± 425.285 vs 499.966 ± 302.273, p =.011). Conclusion: Our findings suggest that ROS generation participates in SLE pathogenesis. The identification of Dicer gene SNP rs3742330 as a potential modifier of SLE risk via mediating IL-6 overproduction suggests a potential avenue for targeted interventions to manage SLE and its associated immune dysregulation. [ABSTRACT FROM AUTHOR]

Background: Downregulated expression of cold-inducible RNA binding protein (CIRP), a stress-response protein, has been demonstrated in the hearts of patients with heart failure (HF). However, whether CIRP plays a critical role in the pathogenesis of HF remains unknown. Zr17-2 is a recently identified CIRP agonist, which can enhance the expression of CIRP in hearts. Herein, we evaluated the effects of zr17-2 on the development of HF in a rat model of myocardial infarction (MI). Methods: Male SD rats were pretreated with CIRP agonist zr17-2 or vehicle saline for 6 consecutive days, followed by MI induction. 1-week post-MI, cardiac function, and structural and molecular changes were determined by echocardiography and molecular biology methods. Results: Excitingly, we found that pretreatment with zr17-2 significantly attenuated MI-induced cardiac dysfunction and dilation, coupled with reduced infarction size and cardiac remodeling. In addition, increased inflammatory response in the peri-infarcted heart including macrophage infiltration and the expression of inflammatory genes were all significantly decreased by zr17-2 pretreatment, suggesting an anti-inflammatory effect of zr17-2. Moreover, zr17-2 pretreatment also upregulated the antioxidant genes (e.g. NQO-1, Nrf2, and HO-1) level in the hearts. In isolated cultured cardiomyocytes, pretreatment with zr17-2 markedly attenuated cell injury and apoptosis induced by oxidative injury, along with elevation of Nrf2-related antioxidant genes and CIRP. However, silencing CIRP abolished zr17-2's antioxidant effects against oxidative injury, confirming that zr17-2's role is dependent on CIRP. Conclusion: Collectively, our study suggests CIRP plays a crucial role in the development of HF and a beneficial effect of CIRP agonist in preventing MI-induced HF, possibly via anti-inflammatory and anti-oxidant pathways. [ABSTRACT FROM AUTHOR]

Single cell chromatin accessibility profiling and transcriptome sequencing are the most widely used technologies for single-cell genomics. Here, we present Microwell-seq3, a high-throughput and facile platform for high-sensitivity single-nucleus chromatin accessibility or full-length transcriptome profiling. The method combines a preindexing strategy and a penetrable chip-in-a-tube for single nucleus loading and DNA amplification and therefore does not require specialized equipment. We used Microwell-seq3 to profile chromatin accessibility in more than 200,000 single nuclei and the full-length transcriptome in ~50,000 nuclei from multiple adult mouse tissues. Compared with the existing polyadenylated transcript capture methods, integrative analysis of cell type-specific regulatory elements and total RNA expression uncovered comprehensive cell type heterogeneity in the brain. Gene regulatory networks based on chromatin accessibility profiling provided an improved cell type communication model. Finally, we demonstrated that Microwell-seq3 can identify malignant cells and their specific regulons in spontaneous lung tumors of aged mice. We envision a broad application of Microwell-seq3 in many areas of research. [ABSTRACT FROM AUTHOR]

Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late‐stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes‐rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles‐rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early‐stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late‐stage PDAC patients undergoing chemotherapy, lower GPC1 tMV‐mProtein and Exo‐mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis. [ABSTRACT FROM AUTHOR]

Background: Oral microbial dysbiosis contributes to the development of oral squamous cell carcinoma (OSCC). Our previous study showed that Prevotella intermedia (P. intermedia) were enriched in the oral mucosal surface, plaque, and saliva of patients with OSCC. Intratumoral microbiome could reshape the immune system and influence the development of various tumors. However, the invasion status of human OSCC tissues by P. intermedia and the pathway through which intratumoral P. intermedia potentiates tumor progression remain unexplored. Methods: P. intermedia in human OSCC or normal tissues was detected by FISH. A mouse OSCC cell line SCC7 was adopted to investigate the effects of heat-killed P. intermedia treatment on cell proliferation, invasion, and cytokine release by using CCK-8 assay, transwell invasion assay and ELISA. Moreover, we established a mouse transplanted tumor model by using SCC7 cells, injected heat-killed P. intermedia into tumor tissues, and investigated the effects of heat-killed P. intermedia on tumor growth, invasion, cytokine levels, immune cell infiltrations, and expression levels by using gross observation, H&E staining, ELISA, immunohistochemistry, mRNA sequencing, and transcriptomic analysis. Results: Our results indicated that P. intermedia were abundant in OSCC and surrounding muscle tissues. Heat-killed P. intermedia promoted SCC7 cell proliferation, invasion and proinflammatory cytokine secretions, accelerated transplanted tumor growth in mice, exacerbate muscle and perineural invasion of OSCC, elevated the serum levels of IL-17A, IL-6, TNF-α, IFN-γ, and PD-L1, induced Treg cells M2 type macrophages in mouse transplanted tumors. The data of transcriptomic analysis revealed that heat-killed P. intermedia increased the expression levels of inflammatory cytokines and chemokines while reduced the expression levels of some tumor suppressor genes in mouse transplanted tumors. Additionally, IL-17 signaling pathway was upregulated whereas GABAergic system was downregulated by heat-killed P. intermedia treatment. Conclusions: Taken together, our results suggest that P. intermedia could inhibit the expression of tumor suppressors, alter the tumor microenvironment, and promote the progression of OSCC. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to explore the relationships between single-nucleotide polymorphisms (SNPs) of crucial molecules in the cGAS-STING signalling pathway and the susceptibility to, induction chemotherapy response of, and prognosis of acute myeloid leukaemia (AML) in Chinese patients. Methods: Thirteen SNPs of crucial molecules in the cGAS-STING signalling pathway were genotyped in 262 AML patients using the Sequenom MassARRAY system. The associations of SNPs with susceptibility, and induction chemotherapy response were analysed using the chi-square test or Fisher's exact test and univariate binary logistic regression, the connection of SNPs with prognosis of AML was analysed using the log-rank test, and Kaplan–Meier curves were applied for survival estimation. Results: In our study, gene polymorphisms of cGAS-STING signalling pathway molecules could be vitally associated with AML. In the recessive model, the cGAS rs311678 gene polymorphism could be closely related to AML susceptibility (CC vs. TT + TC, odds ratio (OR) = 0.480, 95% confidence interval (CI) = 0.260–0.889, p = 0.020). Moreover, IKKA rs3808917 might be associated with the WBC count, cGAS rs311678 could be associated with the bone marrow (BM) blast percentage, and NF-κB rs1056890 under codominant and recessive models could be connected with the HGB level. Patients who were STING rs7380272 TT/CT carriers was likely to have higher insensitivity to induction chemotherapy than CC carriers (TT + CT vs. CC, OR = 2.917, 95% CI = 1.073–7.929, p = 0.036). Survival analysis indicated that the IKKB rs3747811 TT genotype might be associated with decreased overall survival (OS) (p < 0.05). Conclusions: SNPs of molecules in the cGAS-STING signalling pathway could be significantly associated with AML. The cGAS rs311678 gene polymorphism could be associated with AML susceptibility, the STING rs7380272 variant might be related to induction chemotherapy response, and IKKB rs3747811 tended to be associated with AML overall survival. Moreover, IKKA rs3808917 could be associated with the WBC count, cGAS rs311678 could be associated with the BM blast percentage, and NF-κB rs1056890 might be related to the HGB level. [ABSTRACT FROM AUTHOR]

Histone acetylation and methylation, governed by various histone modification (HM) gene families, are vital for plant biological processes. However, there are limited studies that have explored HMs in ornamental horticultural trees, including sweet osmanthus (Osmanthus fragrans). We performed genome-wide search and identified 208 OfHMs, encompassing 81 histone methyltransferases (OfHMTs), 51 histone demethylases (OfHDMs), 49 histone acetyltransferases (OfHATs) and 27 histone deacetylases (HDACs). Our comprehensive analysis covered chromosome locations, gene structures, conserved domains, cis-acting elements, phylogenetic comparisons, protein interaction networks and functional enrichment pathways for these gene families. Additionally, tandem and fragment replications were unveiled as contributors to the expansion of OfHMs, with some genes exhibiting positive selection. Furthermore, we examined OfHM expression profiles across various tissues and flowering stages, and under 5′-azacytidine (Aza) and ethylene treatments. Most OfHMs displayed heightened expression in leaves, and were downregulated during the flower opening and senescence stages, including OfPRMTs, OfHDTs, OfHDAs, OfSRTs, OfJMJs and OfHAGs; 75.86% and 80.77% of the differentially expressed OfHMs were upregulated after Aza and ethylene treatments, including OfHAGs, OfHDAs and OfSDGs. This study offers a comprehensive analysis of the OfHM gene family, which indicated their potential involvement in ethylene and Aza responses, and in the flowering process. These findings provide valuable insights into the role of OfHMs in flowering and stress responses. [ABSTRACT FROM AUTHOR]

The interactions between the rumen microbiota and the host are crucial for the digestive and absorptive processes of ruminants, and they are heavily influenced by the climatic conditions of their habitat. Owing to the harsh conditions of the high-altitude habitat, little is known about how ruminants regulate the host transcriptome and the composition of their rumen microbiota. Using the model species of goats, we examined the variations in the rumen microbiota, transcriptome regulation, and climate of the environment between high altitude (Lhasa, Xizang; 3650 m) and low altitude (Chengdu, Sichuan, China; 500 m) goats. The results of 16 S rRNA sequencing revealed variations in the abundance, diversity, and composition of rumen microbiota. Papillibacter, Quinella, and Saccharofermentans were chosen as potential microbes for the adaptation of Xizang goats to the harsh climate of the plateau by the Spearman correlation study of climate and microbiota. Based on rumen transcriptome sequencing analysis, 244 genes were found to be differentially expressed between Xizang goats and low-altitude goats, with 127 genes showing up-regulation and 117 genes showing down-regulation. SLC26A9, GPX3, ARRDC4, and COX1 were identified as potential candidates for plateau adaptation in Xizang goats. Moreover, the metabolism of fatty acids, arachidonic acids, pathway involving cytokines and their receptors could be essential for adaptation to plateau hypoxia and cold endurance. The expression of GPX3, a gene linked to plateau acclimatization in Xizang goats, was linked to the abundance of Anaerovibrio, and the expression of SLC26A9 was linked to the quantity of Selenomonas, according to ruminal microbiota and host Spearman correlation analysis. Our findings imply that in order to adapt harsh plateau conditions, Xizang goats have evolved to maximize digestion and absorption as well as to have a rumen microbiota suitable for the composition of their diet. [ABSTRACT FROM AUTHOR]

MYB transcription factors play a key role in anthocyanin biosynthesis. However, there are few reports on MYB transcription factors that can regulate anthocyanin biosynthesis in potato. In this study, the StMYB113 gene was isolated from potato and characterized. Transient activation experiments showed that the StMYB113 gene could promote anthocyanin biosynthesis in tobacco leaves by enhancing the expression of genes encoding anthocyanin biosynthesis-related enzymes. In addition, overexpression of the StMYB113 gene promoted anthocyanin accumulation in potato tuber skin. Furthermore, transient luciferase analysis showed that StMYB113 enhanced the promoter activities of StF3H and StDFR genes significantly. These results indicated that the StMYB113 gene could promote anthocyanin accumulation in potato tuber skin by directly activating the expression of StF3H and StDFR. The promoter activity of StMYB113 was significantly higher in light than in dark conditions, suggesting that the expression of StMYB113 is induced by light. These findings illustrate the role and regulation of the StMYB113 gene in potato anthocyanin biosynthesis and enrich the regulatory theory of potato anthocyanin biosynthesis. [ABSTRACT FROM AUTHOR]

Summary: R‐loops, three‐stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single‐stranded DNA, play critical roles in gene expression and genome stability. How R‐loop homeostasis is integrated into chloroplast gene expression remains largely unknown.We found an unexpected function of FtsHi1, an inner envelope membrane‐bound AAA‐ATPase in chloroplast R‐loop homeostasis of Arabidopsis thaliana.Previously, this protein was shown to function as a component of the import motor complex for nuclear‐encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP‐dependent helicase that efficiently unwinds both DNA–DNA and DNA–RNA duplexes, thereby preventing R‐loop accumulation. Over‐accumulation of R‐loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear‐ and chloroplast‐encoded subunits of multi‐protein photosynthetic complexes.This study suggests a mechanical link between protein import and R‐loop homeostasis in chloroplasts of higher plants. [ABSTRACT FROM AUTHOR]

Cholesterol metabolism is crucial for cell survival and cancer progression. The prognostic patterns of genes linked to cholesterol metabolism (CMAGs) in CESC, however, have received very little attention in research. From public databases, TCGA‐CESC cohorts with mRNA expression patterns and the accompanying clinical information of patients were gathered. Consensus clustering was used to find the molecular subtype connected to cholesterol metabolism. In the TCGA‐CESC cohort, a predictive risk model with 28 CMAGs was created using Lasso‐Cox regression. The function enrichment analysis between groups with high‐and low‐risk were investigated by employing GO, KEGG, and GSVA software. The immune cell infiltration was analyzed using ESTIMATE, CIBERSORT, and MCPCOUNTER methods. Finally, we select 7 genes in risk model for further multivariate Cox analysis, and ultimately a hub gene, CHIT1, was identified. Meanwhile, the function of CHIT1 was preliminarily verified in cell and mice tumor model. In conclusion, the abundance of the CHIT1 gene might be beneficial for forecasting the prognosis of CESC, demonstrating that cholesterol metabolism could be a promising treatment target for CESC. [ABSTRACT FROM AUTHOR]

Electromagnetic fields (EMFs) have received widespread attention as effective, noninvasive, and safe therapies across a range of clinical applications for bone disorders. However, due to the various frequencies of devices, their effects on tissues/cells are vary, which has been a bottleneck in understanding the effects of EMFs on bone tissue. Here, we developed an in vivo model system using zebrafish scales to investigate the effects of extremely low-frequency EMFs (ELF-EMFs) on fracture healing. Exposure to 10 millitesla (mT) of ELF-EMFs at 60 Hz increased the number of both osteoblasts and osteoclasts in the fractured scale, whereas 3 or 30 mT did not. Gene expression analysis revealed that exposure to 10 mT ELF-EMFs upregulated wnt10b and Wnt target genes in the fractured scale. Moreover, ß-catenin expression was enhanced by ELF-EMFs predominantly at the fracture site of the zebrafish scale. Inhibition of Wnt/ß-catenin signaling by IWR-1-endo treatment reduced both osteoblasts and osteoclasts in the fractured scale exposed to ELF-EMFs. These results suggest that ELF-EMFs promote both osteoblast and osteoclast activity through activation of Wnt/ß-catenin signaling in fracture healing. Our data provide in vivo evidence that ELF-EMFs generated with a widely used commercial AC power supply have a facilitative effect on fracture healing. [ABSTRACT FROM AUTHOR]

SUMMARY: During the oolong tea withering process, abiotic stresses induce significant changes in the content of various flavor substances and jasmonic acid (JA). However, the changes in chromatin accessibility during withering and their potential impact remain poorly understood. By integrating ATAC‐seq, RNA‐seq, metabolite, and hormone assays, we characterized the withering treatment‐induced changes in chromatin accessibility, gene expression levels, important metabolite contents, and JA and JA‐ILE contents. Additionally, we analyzed the effects of chromatin accessibility alterations on gene expression changes, content changes of important flavor substances, and JA hyperaccumulation. Our analysis identified a total of 3451 open‐ and 13 426 close‐differentially accessible chromatin regions (DACRs) under withering treatment. Our findings indicate that close‐DACRs‐mediated down‐regulated differentially expressed genes (DEGs) resulted in the reduced accumulation of multiple catechins during withering, whereas open‐DACRs‐mediated up‐regulated DEGs contributed to the increased accumulation of important terpenoids, JA, JA‐ILE and short‐chain C5/C6 volatiles. We further highlighted important DACRs‐mediated DEGs associated with the synthesis of catechins, terpenoids, JA and JA and short‐chain C5/C6 volatiles and confirmed the broad effect of close‐DACRs on catechin synthesis involving almost all enzymes in the pathway during withering. Importantly, we identified a novel MYB transcription factor (CsMYB83) regulating catechin synthesis and verified the binding of CsMYB83 in the promoter‐DACRs regions of key catechin synthesis genes using DAP‐seq. Overall, our results not only revealed a landscape of chromatin alters‐mediated transcription, flavor substance and hormone changes under oolong tea withering, but also provided target genes for flavor improvement breeding in tea plant. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is an acute and severe disease transmitted among domestic pigs and wild boars. This disease is notorious for its high mortality rate and has caused great losses to the world's pig industry in the past few years. After infection, pigs can develop symptoms such as high fever, inflammation, and acute hemorrhage, finally leading to death. African swine fever virus (ASFV) is the causal agent of ASF; it is a large DNA virus with 150–200 genes. Elucidating the functions of each gene could provide insightful information for developing prevention and control methods. Herein, to investigate the function of I267L, porcine alveolar macrophages (PAMs) infected with an I267L-deleted ASFV strain (named ∆I267L) and wild-type ASFV for 18 h and 36 h were taken for transcriptome sequencing (RNA-seq). The most distinct different gene that appeared at both 18 hpi (hours post-infection) and 36 hpi was F3; it is the key link between inflammation and coagulation cascades. KEGG analysis (Kyoto encyclopedia of genes and genomes analysis) revealed the complement and coagulation cascades were also significantly affected at 18 hpi. Genes associated with the immune response were also highly enriched with the deletion of I267L. RNA-seq results were validated through RT-qPCR. Further experiments confirmed that ASFV infection could suppress the induction of F3 through TNF-α, while I267L deletion partially impaired this suppression. These results suggest that I267L is a pathogenicity-associated gene that modulates the hemorrhages of ASF by suppressing F3 expression. This study provides new insights into the molecular mechanisms of ASFV pathogenicity and potential targets for ASFV prevention and control. [ABSTRACT FROM AUTHOR]

Ovarian cancer is a complex polygenic disease in which genetic factors play a significant role in disease etiology. A genome-wide association study (GWAS) identified a novel variant on chromosome 9q22.33 as a susceptibility locus for epithelial ovarian cancer (EOC) in the Han Chinese population. However, the underlying mechanism of this genomic region remained unknown. In this study, we conducted a fine-mapping analysis of 130 kb regions, including 1,039 variants in 200 healthy women. Ten variants were selected to evaluate the association with EOC risk in 1,099 EOC cases and 1,591 controls. We identified two variants that were significantly associated with ovarian cancer risk (rs7027650, P = 1.91 × 10-7; rs1889268, P = 3.71 × 10-2). Expression quantitative trait locus (eQTL) analysis found that rs7027650 was significantly correlated with COL15A1 gene expression (P = 0.009). The Luciferase reporter gene assay confirmed that rs7027650 could interact with the promoter region of COL15A1, reducing its activity. An electrophoretic mobility shift assay (EMSA) showed the allele-specific binding capacity of rs7027650. These findings revealed that rs7027650 could be a potential causal variant at 9q22.33 region and may regulate the expression level of COL15A1. This study offered insight into the molecular mechanism behind a potential causal variant that affects the risk of ovarian cancer. [ABSTRACT FROM AUTHOR]

Background: Lipid metabolism plays a pivotal role in asthma pathogenesis. However, a comprehensive analysis of the importance of lipid metabolism-related genes (LMRGs) in regulating the immune microenvironment in asthma remains lacking. The transcriptome matrix was downloaded from the Gene Expression Omnibus (GEO) dataset. Differentially expressed analysis and weighted gene coexpression network analysis (WGCNA) were conducted on the GSE74986 dataset to select hub LMRGs, and gene set enrichment analysis (GSEA) was conducted to explore their biological functions. The CIBERSORT algorithm was used to determine immune infiltration in the asthma and control groups, and the correlation of diagnostic biomarkers and immune cells was performed via Spearman correlation analysis. Subsequently, a competitive endogenous RNA (ceRNA) network was constructed to investigate the hidden molecular mechanism of asthma. The expression levels of the hub genes were further validated in the GSE143192 dataset, and RT‒qPCR and immunofluorescence were performed to verify the reliability of the results in the OVA asthma model. Lastly, the ceRNA network was confirmed by qRT-PCR and RNAi experiments in the characteristic cytokine (IL-13)-induced asthma cellular model. Results: ASAH1, ACER3 and SGPP1 were identified as hub LMRGs and were mainly involved in protein secretion, mTORC1 signaling, and fatty acid metabolism. We found more infiltration of CD8+ T cells, activated NK cells, and monocytes and less M0 macrophage infiltration in the asthma group than in the healthy control group. In addition, ASAH1, ACER3, and SGPP1 were negatively correlated with CD8+ T cells and activated NK cells, but positively correlated with M0 macrophages. Within the ceRNA network, SNHG9-hsa-miR-615-3p-ACER3, hsa-miR-212-5p and hsa-miR-5682 may play crucial roles in asthma pathogenesis. The low expression of ASAH1 and SGPP1 in asthma was also validated in the GSE74075 dataset. After SNHG9 knockdown, miR-615-3p expression was significantly upregulated, while that of ACER3 was significantly downregulated. Conclusion: ASAH1, ACER3 and SGPP1 might be diagnostic biomarkers for asthma, and are associated with increased immune system activation. In addition, SNHG9-hsa-miR-615-3p-ACER3 may be viewed as effective therapeutic targets for asthma. Our findings might provide a novel perspective for future research on asthma. [ABSTRACT FROM AUTHOR]

Objective: To investigate the correlation of maternal PDX1 methylation, NGN3 and Pax6 expression levels with neonatal blood sugars and birth weight in pregnant women with GDM and non GDM. Methods: This was a prospective cohort study. Total 80 pregnant women who were examined and delivered in the Department of Obstetrics of Affiliated Hospital of Hebei University from January 2019 to June 2022 were recruited and divided into two groups according to the results of oral glucose tolerance test (OGTT): the control group and the observation group, with 40 cases in each group. PDXl methylation rate was measured by the methylation-specific PCR method, whereas gene expression levels of PDX1, NGN3 and Pax6 were measured by RT-PCR meanwhile, neonatal blood glucose and hemoglobin A1c (HbA1c) levels were also measured. Results: In comparison with the control group, the observation group had higher levels of FBG, 2-hour postprandial blood glucose (2hPBG) and HbA1c (P<0.05). Neonatal birth weight and insulin levels in the observation group were significantly higher than those in the control group, while Apgar scores and blood glucose were lower than those in the control group(P<0.05). Moreover, the observation group had significantly lower gene expression levels of PDX1, NGN3 and Pax6, and a higher PDX1 methylation rate than the control group (P<0.05). Correlation analysis revealed a negative correlation between neonatal blood glucose levels and PDX1, NGN3 and Pax6 levels in the observation group, with statistical significance (P<0.05). Conclusion: Changes in maternal PDX1 methylation, NGN3 and Pax6 expression levels may lead to abnormal glucose metabolism in neonates, which has a close bearing on neonatal hypoglycemia and blood glucose levels caused by GDM. [ABSTRACT FROM AUTHOR]

Optimal integration of transcriptomics data and associated spatial information is essential towards fully exploiting spatial transcriptomics to dissect tissue heterogeneity and map out inter-cellular communications. We present SEDR, which uses a deep autoencoder coupled with a masked self-supervised learning mechanism to construct a low-dimensional latent representation of gene expression, which is then simultaneously embedded with the corresponding spatial information through a variational graph autoencoder. SEDR achieved higher clustering performance on manually annotated 10 × Visium datasets and better scalability on high-resolution spatial transcriptomics datasets than existing methods. Additionally, we show SEDR's ability to impute and denoise gene expression (URL: https://github.com/JinmiaoChenLab/SEDR/). [ABSTRACT FROM AUTHOR]

Background: Metastasis is the primary cause of recurrence and death in patients with papillary thyroid carcinoma (PTC). LncRNA ACTA2-AS1, a long non-coding RNA, acts as a tumor suppressor in multiple types of human malignancies, while the role of ACTA2-AS1 in PTC metastasis remains unclear. Methods: The ACTA2-AS1 expression in PTC tissues was analyzed. The sponged roles of ACTA2-AS1 via miR-4428/KLF9 axis were identified using starBase tool. The function of ACTA2-AS1 in PTC was performed with in vitro and in vivo experiments. The correlation between DNA methylation and mRNA expressions of these gene in the TCGA dataset was explored. Results: ACTA2-AS1 expression was downregulated in PTC tissues without metastasis and further decreased in PTC tissues with lymph node metastasis compared with that in normal tissues. Functionally, the overexpression of ACTA2-AS1 inhibited the growth, proliferation, and invasion of PTC cells, whereas its depletion exerted opposite effect. In vivo, ACTA2-AS1 expression inhibited PTC metastasis. Furthermore, ACTA2-AS1 acted as a competing endogenous RNA for miR-4428, thereby positively regulating the expression of miR-4428 target gene, KLF9. Finally, miR-4428 overexpression enhanced invasive potential of PTC cells and significantly weakened the effects of ACTA2-AS1 on promotion and inhibition of KLF9 expression as well as invasive ability of PTC cells, respectively. In the TCGA dataset, the methylation level of ACTA2-AS1 was significantly correlated with its mRNA expression (r = 0.21, p = 2.1 × e−6). Conclusions: Our findings demonstrate that ACTA2-AS1 functions as a tumor suppressor in PTC progression at least partly by regulating the miR-4428-dependent expression of KLF9. [ABSTRACT FROM AUTHOR]

Strawberry is a major fruit crop worldwide because its nutritional and health benefits to human health, but its productivity is limited by Botrytis cinerea. Sucrose nonfermentation 1-related protein kinase 1 (SnRK1) has a defense function against pathogens, but the function of SnRK1 in the defense response to B. cinerea in plants is still unclear. In this study, FaSnRK1α-OE and RNAi fruits were constructed and then inoculated with B. cinerea. The result reveals a positive role of FaSnRK1α in the regulation of resistance to gray mold. FaSnRK1α affects SA content by regulating FaPAL1 and FaPAL2 expressions. The genes related to the SA signaling pathway (FaTGA1 and FaTGA2.1) were significantly increased/decreased in FaSnRK1α-OE or FaSnRK1α-RNAi fruit, respectively. FaSnRK1α interacted with the FaWRKY33.2 protein and negatively regulated FaWRKY33.2 expression, and FaWRKY33.2 acts as a repressor of disease resistance to B. cinerea. Finally, FaSnRK1a regulates the expression of six PR genes and the activities of antioxidant enzymes to boost defense response after B. cinerea inoculation. Our findings showed that FaSnRK1a increases the resistance of strawberry fruit to B. cinerea via SA signaling pathway and interaction with the FaWRKY33.2 transcription factor. [ABSTRACT FROM AUTHOR]

• Introducing multiple regulatory factors involved in gene expression during Plasmodium sexual development. • Epigenetic, transcriptional, and post-transcriptional controls of gene expression together contribute to Plasmodium gametocytogenesis and gametogenesis. • Gene expression regulators within Plasmodium sexual development may be the potential ideal target for blocking malaria transmission. Malaria remains one of the most perilous vector-borne infectious diseases for humans globally. Sexual gametocyte represents the exclusive stage at which malaria parasites are transmitted from the vertebrate to the Anopheles host. The feasible and effective approach to prevent malaria transmission is by addressing the sexual developmental processes, that is, gametocytogenesis and gametogenesis. Thus, this review will comprehensively cover advances in the regulation of gene expression surrounding the transmissible stages, including epigenetic, transcriptional, and post-transcriptional control. [ABSTRACT FROM AUTHOR]

Traumatic Brain Injury (TBI) represents a significant public health challenge. Recovery from brain injury necessitates the collaborative efforts of various resident neural cells, predominantly microglia. The present study analyzed rat and mouse RNA expression micro-arrays, high-throughput RNA sequencing and single-cell sequencing data sourced from public databases. To construct an inflammation regulation network around TYRO protein tyrosine kinase-binding protein (TYROBP), to evaluate the role of TYROBP in cell death after TBI. These findings indicate that following TBI, neurons predominantly communicate with one another through the CXC chemokine ligand (CXCL) and CC chemokine ligand (CCL) signaling pathways, employing a paracrine mechanism to activate microglia. These activated microglia intensify the pathological progression of brain injury by releasing factors such as tumor necrosis factor α (TNF-α), vascular endothelial growth factor and transforming growth factor β via the NF-κB pathway. Cells co-culture experiments demonstrated that neurons, impaired by mechanical injury, interact with microglia through non-contact mechanisms. Activated microglia secrete cytokines, including TNF-α, CXCL-8 and CCL2, which trigger an inflammatory response and facilitate neuronal apoptosis. TYROBP gene knockout in microglia was demonstrated to reduce this interaction and reduce neuronal cell apoptosis rates. [ABSTRACT FROM AUTHOR]

Cotton fiber holds immense importance as the primary raw material for the textile industry. Consequently, comprehending the regulatory mechanisms governing fiber development is pivotal for enhancing fiber quality. Our study aimed to construct a regulatory network of competing endogenous RNAs (ceRNAs) and assess the impact of non-coding RNAs on gene expression throughout fiber development. Through whole transcriptome data analysis, we identified differentially expressed genes (DEGs) regulated by non-coding RNA (ncRNA) that were predominantly enriched in phenylpropanoid biosynthesis and the fatty acid elongation pathway. This analysis involved two contrasting phenotypic materials (J02-508 and ZRI015) at five stages of fiber development. Additionally, we conducted a detailed analysis of genes involved in fatty acid elongation, including KCS , KCR , HACD , ECR , and ACOT , to unveil the factors contributing to the variation in fatty acid elongation between J02-508 and ZRI015. Through the integration of histochemical GUS staining, dual luciferase assay experiments, and correlation analysis of expression levels during fiber development stages for lncRNA MSTRG.44818.23 (MST23) and GhKCR2 , we elucidated that MST23 positively regulates GhKCR2 expression in the fatty acid elongation pathway. This identification provides valuable insights into the molecular mechanisms underlying fiber development, emphasizing the intricate interplay between non-coding RNAs and protein-coding genes. • The study investigated the impact of ncRNAs on fiber development by constructing a regulatory network of ceRNAs. • DEGs targeted by ncRNAs were predominantly enriched in phenylpropanoid biosynthesis and fatty acid elongation pathway. • MST23 positively regulates GhKCR2 expression in the fatty acid elongation pathway. • The study provides valuable insights into the molecular mechanisms underlying fiber development, especially in the fatty acid biosynthesis in cotton. [ABSTRACT FROM AUTHOR]

Endogenous immune defenses provide an intrinsic barrier against external entity invasion. Microplastics in the environment, especially those at the nanoscale (nanoplastics or NPs), may pose latent health risks through direct exposure. While links between nanoplastics and inflammatory processes have been established, detailed insights into how they may perturb the innate immune mechanisms remain uncharted. Employing murine and macrophage (RAW264.7) cellular models subjected to polystyrene nanoplastics (PS-NPs), our investigative approach encompassed an array of techniques: Cell Counting Kit-8 assays, flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) fluorescence staining, cell transfection, cell cycle scrutiny, genetic manipulation, messenger RNA expression profiling via quantitative real-time PCR, and protein expression evaluation through western blotting. The results showed that PS-NPs caused RAW264.7 cell apoptosis, leading to cell cycle arrest, and activated the cGAS-STING pathway. This resulted in NF-κB signaling activation and increased pro-inflammatory mediator expression. Importantly, PS-NPs-induced activation of NF-κB and its downstream inflammatory cascade were markedly diminished after the silencing of the STING gene. Our findings highlight the critical role of the cGAS-STING pathway in the immunotoxic effects induced by PS-NPs. We outline a new mechanism whereby nanoplastics may trigger dysregulated innate immune and inflammatory responses via the cGAS/STING pathway. • PS-NPs can activate innate immunity. • 2.PS-NPs can induce inflammatory response through cGAS-STING pathway. • After macrophages knocked down the STING gene, the inflammatory response induced by PS-NPs was inhibited. [ABSTRACT FROM AUTHOR]

Sea cucumber (Apostichopus japonicus) is a commercially important mariculture species in China. Generally, sea cucumbers weighing around 100 g and older than two years, with mature gonads are harvested for food. Sexual dimorphism is widely observed in aquaculture animals, and sex control biotechnologies have created typical cases of genetic breeding in several aquaculture species. Clarifying the sex differences in important economic traits and developing a rapid genetic sex identification method are critical steps for establishing a monosex aquaculture population. In the present study, we established a loop-mediated isothermal amplification (LAMP) based method to identify the genetic sex of sea cucumbers in the field without complex equipment. Subsequently, we investigated the growth difference between males and females in terms of weight gain, and revealed that there was no significant growth difference between males and females 1–2-year-old juvenile sea cucumbers with undifferentiated gonads. However, among 2–3-year-old adult sea cucumbers with differentiated gonads, males exhibited greater growth advantages compared to females. The weight increment and specific growth rate (SGR) of male sea cucumbers were 1.73 and 1.52 times greater than those of female sea cucumbers, respectively, highlighting the economic value in producing all-male populations of A. japonicus. Furthermore, the expression levels of growth trait candidate genes (hgfr1 and c-myc) in the body wall tissues showed no significant differences between males and females of 1–2-year-old A. japonicus. However, in 2–3-year-old A. japonicus , the expression levels of these genes were significantly higher in males than in females. Conversely, cyp1a1 and cyp17a genes were more highly expressed in differentiated ovaries than in differentiated testes. This study provides a solid foundation for achieving sex control breeding in sea cucumbers. • A rapid LAMP-based method was established to identify the genetic sex of A. japonicus • Males exhibit greater growth advantages compared to females among 2–3-year-old adult sea cucumbers • The expression levels of growth trait candidate genes (hgfr1 and c-myc) were significantly higher in males than in females. • Cyp1a1 and cyp17a genes exhibited higher expression in differentiated ovaries compared to differentiated testes. [ABSTRACT FROM AUTHOR]

• miR-196b-5p may act as a critical link in glaucoma pathogenesis, enriching with and negatively regulating Nras at the transcriptional level. • miR-196b-5p is highly expressed in trabecular cells in both animal models of glaucoma and human glaucoma and affects trabecular cell function. • miR-196b-5p promotes the inflammatory process of glaucomatous trabecular cells, inhibits proliferation and migration, and alters their cytoskeletal structure. Glaucoma, an insidious ophthalmic pathology, is typified by an aberrant surge in intraocular pressure (IOP) which culminates in the degeneration of retinal ganglion cells and optical neuropathy. The mitigation of IOP stands as the principal therapeutic strategy to forestall vision loss. The trabecular meshwork's (TM) integrity and functionality are pivotal in modulating aqueous humor egress. Despite their potential significance in glaucomatous pathophysiology, the implications of microRNAs (miRNAs) on TM functionality remain largely enigmatic. Transcriptomic sequencing was employed to delineate the miRNA expression paradigm within the limbal region of rodent glaucoma models, aiming to elucidate miRNA-mediated mechanisms within the glaucomatous milieu. Analytical scrutiny of the sequencing data disclosed 174 miRNAs with altered expression profiles, partitioned into 86 miRNAs with augmented expression and 88 with diminished expression. Notably, miRNAs such as hsa-miR-196b-5p were identified as having substantial expression discrepancies with concomitant statistical robustness, suggesting a potential contributory role in glaucomatous progression. Subsequent in vitro assays affirmed that miR-196b-5p augments the inflammatory cascade within immortalized human TM (iHTM) and glaucoma-induced human TM (GTM3) cells, concurrently attenuating cellular proliferation, motility, and cytoskeletal architecture. Additionally, miR-196b-5p implicates itself in the regulation of IOP and inflammatory processes in rodent models. At a mechanistic level, miR-196b-5p modulates its effects via the targeted repression of Nras (neuroblastoma RAS viral oncogene homolog). Collectively, these transcriptomic investigations furnish a comprehensive vista into the regulatory roles of miRNAs within the glaucomatous framework, and the identification of differentially expressed miRNAs alongside their targets could potentially illuminate novel molecular pathways implicated in glaucoma, thereby aiding in the development of innovative therapeutic avenues. [ABSTRACT FROM AUTHOR]

Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, controls arginine dimethylation of a variety of substrates. While many papers have reported the function of mammalian PRMT5, it remains unclear how PRMT5 functions in chicken cells. In this study, we found that chicken (ch) PRMT5 is widely expressed in a variety of chicken tissues and is distributed in both the cytoplasm and the nucleus. Ectopic expression of chPRMT5 significantly suppresses chIFN-β activation induced by chMDA5. In addition, a prmt5 gene-deficient DF-1 cell line was constructed using CRISPR/Cas9. In comparison with the wild-type cells, the prmt5−/− DF-1 cells displays normal morphology and maintain proliferative capacity. Luciferase reporter assay and overexpression showed that prmt5−/− DF-1 cells had increased IFN-β production. With identified chicken PRMT5 and CRISPR/Cas9 knockout performed in DF-1 cells, we uncovered a functional link of chPRMT5 in suppression of IFN-β production and interferon-stimulated gene expression. [ABSTRACT FROM AUTHOR]

In traditional Chinese herbal medicine, rhubarb is said to remove accumulation with purgation, clearing heat, and discharging fire. Modern pharmacology has shown that rhubarb extract has a purgative effect when given to experimental animals in an appropriate dose. However, the active components and their mechanism of action are still not clearly defined. The current research aimed to evaluate the synergistic stool-softening effects and explore the action mechanism of rhubarb free anthraquinones (RhA) and their monomers on constipation in rats. A rat model of water deficit-induced constipation was established to induce constipation, and these rats were treated with RhA and its monomers. ELISA, histopathology, immunohistochemistry, qPCR and Western blotting based on network pharmacology and molecular docking were conducted to explore the possible mechanism of action of RhA and its monomers. RhA, aloe-emodin, rhein, and chrysophanol showed stool-softening activity, and the combination of aloe-emodin and rhein had the strongest softening effect on faecal pellets. Aloe-emodin, rhein, and chrysophanol significantly increased the serum levels of vasoactive intestinal peptide (VIP), motilin (MTL), and substance P (SP), upregulated the expression of VIP, cyclase-associated protein 1 (CAP1), protein kinase A (PKA), cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 3 (AQP3), aquaporin 4 (AQP4), and aquaporin 8 (AQP8), decreased the expression of epithelial sodium channel (ENaC) and Na+/H+ exchanger 3 (NHE3), and reduced the colonic tissue concentration of Na+-K+-ATPase in the constipated rats. Osmolality of colonic fluid in model rats treated by RhA, aloe-emodin, rhein, and chrysophanol was increased. Aloe-emodin, rhein, and chrysophanol were the stool-softening components of the RhA extract, and there were certain drug-interactions between the components. RhA upregulated VIP expression, activated the cyclic adenosine monophosphate protein kinase A (cAMP/PKA) pathway, and further stimulated CFTR expression while inhibiting NHE3 and ENaC expression, resulting in a hypertonic state in the colonic lumen. Water transport could then be driven by an osmotic gradient, which in turn led to the upregulation of AQP3, AQP4, and AQP8 expression. In addition, RhA likely improved gastrointestinal motility by increasing serum VIP, SP, and MTL concentrations, thus promoting faecal excretion. [Display omitted] • Aloe-emodin, rhein and chrysophanol were the stool-softening components of rhubarb, and there were certain drug-interactions between the components. • Aloe-emodin, rhein and chrysophanol upregulated VIP expression, activated the cAMP/PKA pathway, further stimulated CFTR expression while inhibited NHE3 and ENaC expression, resulting in osmolality driven water transport, which in turn led to upregulation of AQP expression. [ABSTRACT FROM AUTHOR]